Use of enzymes with a wide pH activity range as medicaments for promoting digestion

ABSTRACT

Disclosed is a combination of two or more lipase enzymes, and its use for treating a lipid digestion deficiency and/or a digestive disorder. At least one lipase enzyme has a pH optimum at an acidic pH value, while at least one other lipase enzyme has a pH optimum at an alkalic pH value.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims benefit of and priority under 35 U.S.C. § 111 ofU.S. patent application Ser. No. 15/545,583, now allowed, under 35U.S.C. § 371 of PCT/EP2016/051341, filed Jan. 22, 2016, which claims thebenefit under 35 U.S.C. § 119(e) of Great Britain Application No.1501081.2, filed Jan. 22, 2015, which are incorporated herein byreference in their entirety.

REFERENCE TO SEQUENCE LISTING

The Sequence Listing submitted Nov. 18, 2019 as a text file named“003168_2024 DIV_Sequence-Listing.txt,” created on Nov. 18, 2019, andhaving a size of 12,830 bytes is hereby incorporated by referencepursuant to 37 C.F.R. § 1.52(e)(5).

FIELD OF THE INVENTION

The present invention relates to a medicament containing homologousexpressed enzymes from ciliates for treating digestive disorders.

BACKGROUND

Digestive disorders play an increasingly greater role in the generalmedical and internal medical practice. Such digestive disorders are inmany cases the consequence of a more or less pronounced deficiency inso-called pancreatic enzymes. In a healthy state, these enzymes aresynthesized in the pancreas by highly specialized cells, the so-calledacinic cells, and secreted by exocytosis through juice glands and themain pancreatic duct into the duodenum. The daily amount of pancreaticsecretion is about 2 liters. In addition to fat digesting lipase, thepancreatic secretion also contains enzymes for the digestion of proteins(trypsin, chymotrypsin and carboxypeptidases) and carbohydrates(α-amylase). The secretion of pancreatic enzymes is exactly controlledby endogenous control mechanisms by means of hormones, such as gastrin,secretin and pancreozymin. This control system can be disturbed by alarge number of causes to result in a reduction of pancreatic enzymesecretion or in a complete subsiding of the exocrine pancreaticfunction. This in turn causes that the chyme is not digested in thesmall intestine, and a digestive disorder occurs. This disease of thedigestive tract, which is also referred to as exocrine pancreaticinsufficiency (EPI), can have different causes. In addition to dyspepsiacaused by medicaments, chronic atrophic gastritis and chronicpancreatitis, frequently caused by alcohol consumption, disorders causedby surgery (e.g., Billroth I and II, vagotomy, pancreas resection) andcystic fibrosis are etiologic factors of pancreatic insufficiency. Atany rate, chronic digestive disorders are of considerable social-medicaland thus economic importance, because the symptoms frequently cause thepatients to be nondescript and have a shortened expectation of life.

Pancreatogenic digestive disorders and especially EPI cause a lot ofcomplaints in the patients, such as diarrhea, mass stools, sensations ofrepletion, upper abdominal complaints, weight loss etc.

Irrespective of the causes and the manifestation of pancreatogenicdigestive disorders or EPI to avoid malnutrition related morbidity andmortality, it is pivotal to commence a substitution therapy with enzymesas soon as EPI is diagnosed. This means that the lacking enzymes,predominantly lipase, protease and amylase, but also other enzymes, mustbe supplied externally. In the therapy, the enzymes are taken in orallyby the patient mostly in the middle of the meal and go through thestomach and arrive in the small intestine, where they perform digestionof the chyme and thus adopt the function of the lacking endogenouspancreatic enzymes. The preparations employed must contain a sufficientamount of enzymes. In addition, the enzymes must be provided in anenteric formulation, have a small particle size and be completelybioavailable in the digestive tract. For treating digestive disordersbased on the lacking of pancreatic enzymes often pancreatic enzymereplacement therapy (PERT) based on the substitution/replacement of theleading enzyme lipase and the protease, is used. For PERT a wide varietyof enzyme preparations are already on the market. These are partly basedon pancreatic enzymes from pigs, such as the preparations Combizym®,Festal®, Pankreon®, Kreon®, Panzytrat®, Meteozym® or Enzym-Lefax N®Preparations containing pancreatic enzymes, so called pancreatic enzymeproducts or PEPs, are mostly obtained from pigs from slaughter, forexample, pancreas, of pigs. The final product of the preparation processis pancreatin. PEPs are composed of porcine lipase, amylase, andprotease and are used in patients with EPI secondary to cystic fibrosis,chronic pancreatitis, and pancreatectomy.

In 1938, PEPs were exempted from the Food, Drug, and Cosmetic Act of1938 and never underwent a formal Food and Drug Administration (FDA)review process (Giuliano C A L Dehoome-Smith M L, Kale-Pradhan P B(2011) Pancreatic enzyme products: digesting the changes. AnnPharmacother. 45(5):658-66.

PEPs from pig origin cannot be employed with patients suffering fromdigestive disorders who have a pig protein allergy. In addition, pigsare considered a natural reservoir of human-pathogenic influenza virusesand a vast number of viruses from porcine origin, so that contaminationof pancreatin with such viruses cannot be ruled out. In other words,pancreatic tissue, which would present slaughter waste, if not furtherprocessed, can exhibit a high degree of viral contamination. Inconsequence based on its natural origin, the pancreatic tissue,pancreatin and PEP also can be contaminated with viruses from porcineorigin. It has to be emphasized that the International Conference onHarmonisation of Technical Requirements for Registration ofPharmaceuticals for Human Use (ICH) sets a very high standard in itsguideline ICH Topic Q 5 A (R1) and demands as the best reasonableassurance that the product is free of virus contamination. The Centerfor Drug Evaluation and Research (CDER) of the US FDA already requestedintensive risk mitigation strategies for lipase containing PEPs likeCreon.

This, because there is a risk for contamination of PEPs with PorcineParvovirus and Porcine Circovirus as well as significant number of swineviruses that are known human pathogens.

Unfortunately, neither manufacturer thus far has found any viralinactivation method that can successfully demonstrate acceptable virusclearances of PEP without also degrading or reducing the pancreaticenzymes, particularly lipase, to unacceptable levels. Due to furtherlimitations associated with analytical testing of such a complexbiological material, like pancreatin, it will be difficult to determinewhat degradants may be introduced into the product as a result of anyadded viral clearance steps. In conclusion, process steps that can beeffective against viruses have a high potential for changing the natureof PEPs, thus having a potentially serious impact on the drug's quality,safety and efficacy. However, because there are almost no alternativeresources on the market for lipases than PEPs, they still represent theonly permitted drug compositions for the treatment of EPI.

In addition, for particular groups of patients, a disadvantage of theuse of PEPs is the origin from pig. Usually, the pancreas of pigs isused, which cannot be tolerated by patients of Judaic or Islamicreligion due to religious instructions. Furthermore pancreatin is ahomogenate from the cells of pancreatic tissues (usually from pigs). Dueto the rupture of a large number of acinic cells, it contains, inaddition to pancreatic enzymes, a wide variety of other enzymes andproteins as well as further high and low molecular weight compounds. Thecomposition of pancreatin is due to its industrial preparation process.To obtain pancreatin, pancreas of pigs are deep-frozen as quickly aspossible after slaughtering, collected and broken up mechanically. Forthe stabilization and activation of the enzymes, various additives areadded to the homogenate. This is followed by defatting with organicsolvents, such as acetone, the removal of fibrous substances, anddewatering and drying by lyophilization. In view of the problemsassociated with the management of organic solvents and the coststhereof, there thus remains a need for a method of enzyme productionwhich minimises the use of organic solvents or work without of organicsolvents. For the preparation of particular dosage forms, furthergalenic processing may be effected into micropellets, tablets, capsules,pastes, creams, gels, oils or other formulations. Frequently, pancreatinbased PEPs are mixed with various support materials and buffersubstances. Further, granulated pancreatin is coated with acid-stablefilms or lacquers for protection against the low pH value of humangastric juice. The two latter processing steps are to ensure that theacid-labile PEPs can fulfill their digestive function at the targetsite, the duodenum (small intestine), but prevent the enzymes from beingactive in the acidic upper Duodenum of many pancreatitis Patients.

A newer PEP, which contains lipase, is Zenpep®. Zenpep® is a combinationof porcine-derived lipases, proteases and amylases from pigs forslaughter indicated for the treatment of EPI due to cystic fibrosis orother conditions. In opposite to common marketed PEPs for EPI, Zenpep®does not show great variability in the amount of enzymes included ineach capsule. The variability of the common PEPs is due in part to themanufacturer practice of overfilling capsules to account for enzymedegradation that occurs over the course of the product's shelf life.Variability in the product's enzyme content can lead to inconsistenttherapeutic effects by either providing too much or too little of therequired enzymes, which may lead to the suboptimal treatment of thepatient's EPI. In addition, overfilled products may increase the risk offibrosing colonopathy, which has been associated in some reports withlong-term exposure to high-dose PERT. These problems can be avoided bythe application of Zenpep®. However, Zenpep® contains the same porcinepancreatic lipase like common PEPs. In consequence porcine pancreaticlipase in Zenpep® becomes efficient only in the presence of porcinecolipase in the duodenum. Thus, Zenpep® also nearly an unpurifiedprotein mixture from pigs for slaughter, because purification in themanufacturing process would lead to a potential loss of the colipase.Additionally, Zenpep® can also contain viral contamination and, becauseof the necessary precipitation and defattening steps, residual organicsolvents as described for PEPs. In consequence, as for common PEPs forPERT, porcine pancreatic lipase in this composition can be contaminatedwith viruses and/or organic solvents.

In order to circumvent the use of pig pancreatic tissue as source forlipase and protease, some researchers suggested the use of enzymes fromfish or other marine animals generally described in FR No. 1015566, aswell as compositions of enzymes from the gastrointestinal tract of krill(crustaceans from the class Euphausiaceae) and capelin fish (U.S. Pat.No. 4,695,457). However these natural resources are difficult to handleand to process for characterized enzyme preparations in industrial scaleand in consequence enzyme preparation containing lipases from theseorigins never reached the market.

Due to the problems of contamination with viruses and organic solventscharacterizing conventional enzyme preparation from pig pancreatictissue, the use of microbially-derived enzymes as alternatives toporcine-derived PEP has been proposed. For example, U.S. Pat. No.6,051,220 describes compositions comprising one or more acid stablelipases and one or more acid stable amylases, both preferably of fungalorigin. United States patent application 2004/0057944 describescompositions comprising Rhizopus delemar lipase, Aspergillus melleusprotease and Aspergillus oryzae amylase.

In part, some enzyme preparations therefore also contain microbialenzymes from mold extracts, such as Nortase® and Combizym®. In spite oftheir resistance to acid and although they do not depend on colipase,their clinical efficacy is low due to rapid intraluminal inactivation bybile salts and proteases.

A recombinant enzyme preparation in development, which contains a bilesalt stable lipase, is a drug with the brand name Kiobrina, which is arecombinant human bile-salt-stimulated lipase (rhBSSL). This rhBSSLshall improve the digestion and absorption of essential fatty acids,such as long-chain poly-unsaturated fatty acids but also cholesterolesters in the human gut. rhBSSL is expressed and produced in mammaliancells and is developed by Sobi for enzyme therapy to improve growth anddevelopment in preterm infants receiving pasteurized breast milk and/orformula. The rationale for substitution of rhBSSL in pasteurized breastmilk or infant formula is to restore the natural lipase activity levelthat is either lost on pasteurization or totally absent in formula. Adisadvantage is, that the enzyme is active only in the presence ofprimary bile salts, which are often insoluble and, thus, not availablein the duodenum, especially under the condition of exocrine pancreaticinsufficiency. Furthermore the developing company Sobi announced thatclinical data show that the rBSSL did not meet its primary endpoint and,thus, the rhBSSL is does not fulfill its function for enzymesubstitution therapy in the human gastrointestinal tract.

Another recombinant lipase for the treatment of exocrine pancreaticinsufficiency is the crystallized, purified cross-linkedPseudomonas/Burkholderia cepacia lipase with of molecular weight of 30kDa, which is part of an enzyme composition namedALTU-135/TheraCLEC®/Trizytek®/Liprotamase®)/Sollpura®) (now with thebrand name Sollpura®) developed by Anthera Pharmaceuticals and which isalso disclosed in U.S. Pat. No. 7,718,169.

This microbial lipase is of bacterial origin, which is described inUnited States patent application 2001/0046493, can be produced byrecombinant DNA technology. The enzyme is secreted in the culturemedium, and requires a complex production process for purification andcrystallization in order to stabilize the enzyme for administration inthe human gastro intestinal system. After crystallization, the lipasecrystals have to be chemically and covalently cross-linked in orderreach sufficient acid stability. In vitro studies suggest that thismodification called cross-linked enzyme crystals (CLEC) processincreases stability of the lipase and that the cross-linked lipase isinsoluble at acidic pH representative of the stomach. However, it hasbeen shown that diffusion effects have been a serious problem for thepractical use of crystalline enzymes (Alter, G. M., Leussing, D. L.,Neurath, H. Bert L. Vallee, B. L., 1977) Kinetic Properties ofcarpoxipeptidase B in solution and crystals. Biochemistry 16 (16):3663-3668). Furthermore it has been shown, that CLEC lipases show asignificantly reduced enzyme activity on certain substrates compared tosoluble enzymes (Margolin, A. L. (1995) Novel crystalline catalysts.Trends in Biotechnology 14 (7): 223-230). Moreover, thePseudomonas/Burkholderia cepacia lipase has a broad pH optimum from pH4-8 with a strong decline of activity for pH values larger than 8.However, Pseudomonas/Burkholderia cepacia lipase is not active under pHconditions significantly larger than pH 8. In consequence this enzymecannot support the digestion of lipids under strong alkaline conditions(larger than pH 8.5 or higher). One other problem forPseudomonas/Burkholderia cepacia lipase is, that the enzyme is a proteinfrom a pathogenic bacterium. Pseudomonas/Burkholderia cepacia(explanation: formerly known as Pseudomonas cepacia, the bacterium nowis known as Burkholderia cepacia) is an important human pathogen whichmost often causes pneumonia in immunocompromised individuals withunderlying lung disease such as cystic fibrosis or chronic granulomatousdisease.

Patients with cystic fibrosis are at risk for acquiring the well known“Burkholderia cepacia syndrome and in consequence the so called“Burkholderia cepacia syndrome” is a serious condition in patients withcystic fibrosis that does not always respond well to treatment.

In the case of such a Pseudomonas/Burkholderia cepacia infectionlong-term active memory is acquired following infection by activation ofB and T cells, while some of their offspring become long-lived memorycells. Throughout the lifetime of human, these memory cells as part ofthe “adaptive immune system”, remember each specific pathogenencountered and can mount a strong response if the pathogen is detectedagain.

Under the conditions of oral treatment with Pseudomonas/Burkholderiacepacia lipase the oral uptake of enzymes from Pseudomonas/Burkholderiacepacia as part of enzyme replacement therapy (in the case of cysticfibrosis) represents an incorporation of exogenous antigens. Theantigens will be detected by the adaptive immune system as pathogens andwould result 1. in the activation of the immune system (so calledimmunological memory), 2. the generation of antibodies against theenzymes and 3. the risk of an exaggerated immune response in theintestine mucosa including potential allergic and autoimmune reactionsand sepsis.

This risk of an of exaggerated immune response in the case of amedication of cystic fibrosis with Pseudomonas/Burkholderia cepaciaenzymes would be counterproductive for the treatment of patients andwould exacerbate the patients' health situation.

The combination of insufficient efficacy and the risk of the abovedescribed detrimental side effects have already raised serious concernof regulatory agencies. In 2011, the Gastrointestinal Drugs AdvisoryCommittee of the Food and Drug Administration (FDA) of the US Departmentof Health and Human Services rejected the finding that Sollpura®'s (theenzyme preparation with Pseudomonas/Burkholderia cepacia lipase)benefits outweighed its risks, arguing that additional efficacy datawere needed before it could conclude that it worked in patients betterthan existing porcine derived pancreatic enzyme products.

Finally it has been shown that there is substantial evidence thatSollpure is less efficacious than the porcine-derived PEPs and appearsto expose patients with EPI to greater risk (Carome M. Wolfe S.Testimony to the FDA Gastrointestinal Drug Advisory Committee regardingliprotamase—risk: benefit assessment; ethics of further clinical trials.Washington D.C.: Public Citizen Research Group. Jan. 12, 2011)

Moreover, the U.S. Pat. No. 5,998,189 discloses a recombinant acidstable dog gastric lipase produced in E. coli and claims the expressionof acid stable dog gastric lipase in E. coli as well as in otherprokaryotic and eukaryotic expression systems. Additionally, theexpression and extraction of this acid stable dog gastric lipase intransgenic corn has been demonstrated Zhong, Q., GU, Gu, Z. and Glatz,C. E. (2006) Extraction of recombinant dog gastric lipase fromtransgenic corn seed. J. Agric. Food Chem. 54: 8086-8092).

However, dog gastric lipase shows a very low pH optimum at pH 4 with avery narrow pH profile from pH 3-pH 5 (Carriere, F., Moreau, H., Raphel,V., Laugier, R., Benicourt, C., Junien, J.-L. and Verger, R., 1991)Purification and biochemical characterization of dog gastric lipase.Eur. J. Biochem. 202: 75-83)

The goal of an enzyme preparation containing lipase displaying thehighest efficacy at the lowest dose, and characterized by a well-definedsafety profile, remains of great importance to all patients sufferingfrom pancreatic insufficiency, including those in the cystic fibrosiscommunity.

TABLE 1 Summary enzyme preparation for PERT approaches pH name and brandoptimum name of on market source enzymes lipase remarks limitations PEPs(Creon®, Pancreatin pancreatic enzymes from pig 7 to 8 nobiotechnological Cotazyme®, or process, no Ultrase®, Viekase® pancreaticcontamination etc.) extract process, so drug contamination with productsviruses and bacteria from pig are possible due to pancreasnon-sterilized tissue tissue from slaughter house Zenpep® Pancreatinpancreatic enzymes from pig with 7 to 8 no biotechnological or definedenzyme activity process, no pancreatic containment extract process, sodrug contamination with products viruses and bacteria from pig arepossible due to pancreas non-sterilized tissue tissue from slaughterhouse no product tissue lypolytic and proteolytic enzymes no pH nobiotechnological from from gastrointestinal tract optimum process, nokrill and disclosed containment capelin process, so fish contaminationwith viruses and bacteria are possible due to non-sterilized tissue withwaste character of the natural source Nortas®/Combizym® Microbial-Rhizopus oryzae/delemar lipase, 6.6 to Rhizopus mould Aspergillusmelleus protease and 7.5 oryzae/delemar is an fungus Aspergillus oryzaeamylase opportunistic human pathogen, potential immune reaction ifprevious Pseudomonas/ Burkholderia cepacia infection, lipase unstableunder physiological bile salt concentrations Kiobrina® recombinant humanbile salt stable 7.3 to complex and lipase (MBSSL) 8.6 expensiverecombinant production process in animal cell lines ALTU- Microbial-Recombinant 8.5 to cross- Pseudomonas/Burkh 135/TheraCLEC®/ culturePseudomonas/Burkholderia 9 linked olderia cepacia Trizyt® of E. colicepacia lipase enzyme opportunistic human ek®/Liprotamase®/ bacteriacrystals Pathogen potential Sollpura® (CLEC immune reaction if process)previous Pseudomonas/ Burkholderia cepacia infection, low activity underacidic conditions no product Microbial- acid stable dog gastric lipase 3to 5 low activity under culture produced in E. coli alkaline conditionsof E. coli (http://www.google.com/patients/ bacteria U.S. Pat. No.5,998,189) Merispase® transgenic acid stable dog gastric lipase in 3 to4 low activity under corn transgenic corn alkaline conditions no productMicrobial- acid stable Tetrahymena lipases 3.5 to low activity underculture 4.5 alkaline conditions of T. therrnophila

In most patients, lipid digestion cannot be completely normalized bycurrent standard therapy. Furthermore the production of lipases forhuman administration is cumbersome and expensive. The instant inventionaddresses these issues.

SUMMARY OF THE INVENTION

The present invention provides a medicament containing homologousexpressed enzymes from ciliates for treating digestive disorders. Theinvention and general advantages of its features will be discussed indetail below.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1-3 show the lipolytic activity of three different overexpressedTetrahymena lipases at different pH values.

FIG. 4 shows the stability of two lipases in human gastric fluid.

FIG. 5 shows the lipolytic activity of a lipase in the presence ofvarious amounts of a physiologic mixture of bile acids.

FIG. 6 A shows a donor vector and FIG. 6B shows an acceptor vector usedin the context of the present invention.

FIGS. 7A, 7B, and 7C show CL activity. A) Lipolysis of pig fat.Activated and heat inactivated CL was incubated with fat at 37° C. B)Specific activity of CL and standard enzyme at different pH values. Thelipolytic activity was determined by pH-STAT and pH-endpoint titrationsin an olive oil-water-emulsion in the presence of bile salt(Na-taurocholat) at 37° C. C) Activity of CL and standard enzyme atdifferent temperatures. The lipolytic activity was determined by pH-STATand pH-endpoint titrations in an olive oil-water-emulsion in thepresence of bile salt (Na-taurocholat) at temperatures between 5-60° C.

DETAILED DESCRIPTION OF THE INVENTION

Before the invention is described in detail, it is to be understood thatthis invention is not limited to the particular component parts of thedevices described or process steps of the methods described as suchdevices and methods may vary. It is also to be understood that theterminology used herein is for purposes of describing particularembodiments only, and is not intended to be limiting. It must be notedthat, as used in the specification and the appended claims, the singularforms “a”, “an”, and “the” include singular and/or plural referentsunless the context clearly dictates otherwise. It is moreover to beunderstood that, in case parameter ranges are given which are delimitedby numeric values, the ranges are deemed to include these limitationvalues.

It is further to be understood that embodiments disclosed herein are notmeant to be understood as individual embodiments which would not relateto one another. Features discussed with one embodiment are meant to bedisclosed also in connection with other embodiments shown herein. If, inone case, a specific feature is not disclosed with one embodiment, butwith another, the skilled person would understand that does notnecessarily mean that said feature is not meant to be disclosed withsaid other embodiment. The skilled person would understand that it isthe gist of this application to disclose said feature also for the otherembodiment, but that just for purposes of clarity and to keep thespecification in a manageable volume this has not been done.

Furthermore, the content of the prior art documents referred to hereinis incorporated by reference. This refers, particularly, for prior artdocuments that disclose standard or routine methods. In that case, theincorporation by reference has mainly the purpose to provide sufficientenabling disclosure, and avoid lengthy repetitions.

According to one aspect of the invention, a combination of two or morelipase enzymes is provided, wherein at least one lipase enzyme has a pHoptimum at an acidic pH value, while at least one other lipase enzymehas a pH optimum at an alkalic pH value.

One way to define the pH optimum is the pH range in which the lipaseas >50% of its peak activity, as determined with, e.g., the Nixon Testor the titration test (see below). The term “pH optimum” is usedsynonymously with the term “maximum lipolytic activity”.

The term “acidic pH value” means a pH between ≥0 and ≤7, while the term“alkalic pH value” means a pH between > and ≤14.

The term “lipase enzyme”, as used herein, refers to an enzyme thatcatalyzes the hydrolysis of fats (lipids). Lipases are a subclass of theesterases and perform essential roles in the digestion, transport andprocessing of dietary lipids (e.g. triglycerides, fats, oils) in most,if not all, living organisms. The term lipases encompass the followingsubtypes: bile salt-dependent lipase, pancreatic lipase, lysosomallipase, hepatic lipase, lipoprotein lipase, hormone-sensitive lipase,gastric lipase, endothelial lipase, pancreatic lipase related protein 2,pancreatic lipase related protein 1 and lingual lipase

The inventors have surprisingly found that a combination of these two ormore lipase enzymes wherein one of which has a pH optimum at an acidicpH value, while at least one other has a pH optimum at an alkalic pHvalue, significantly increases the efficacy of a lipase therapy.

According to one embodiment, the lipolytic activity of at least onelipase enzyme is determined with the Nixon Test or the titration test.

The Nixon test is disclosed in Nixon & Chang 1979, content of which isincorporated herein. Details of this test are disclosed elsewhereherein. The titration test is disclosed in United States Pharmacopeia23, NF18 1095, pp 1150-1151, content of which is incorporated herein.

In one embodiment, the at least one lipase enzyme is a lipase enzymeencoded, expressed and/or produced by an organism of the order ciliates.

In one embodiment, said ciliate is from the family Tetrahymenidae. Morepreferably, said ciliate is from the genus Tetrahymena. Most preferably,said ciliate is from the Tetrahymena thermophile. A ciliate based lipaseproduction system provides an economical, simple and reliable method forthe production of lipases, which have a drastically increased specificactivity compared to the available competitors and thus a highlyenhanced therapeutic potential.

Since no viruses have been found in Tetrahymena combined with the greatevolutionary distance between mammalians and ciliates the safety of theproduct is expected to be much higher, while the production can be runwith more stability and less risk of failure due to viral infections.

In one embodiment, at least one lipase enzyme is a lipase enzymeaccording to claim 3 which has been modified by site directed or randommutagenesis and subsequent selection.

In one embodiment, one lipase enzyme has a pH optimum at a pH valuewhich occurs in the stomach of a mammal, while at least one other lipaseenzyme has a pH optimum at a pH value which occurs in the lower smallintestine of a mammal.

The intraluminal pH of a mammalian gastrointestinal tract including thestomach is discussed in Fallingborg J, Dan Med Bull. 1999 June;46(3):183-96. Some typical values for human gastrointestinal tract areshown in the following table:

TABLE 2 Stomach pH 1-4 Duodenum pH 6 terminal ileum pH 7.4 caecum pH 5.7

Thus, due to it's broad pH spectrum, the product promotes lipolysis overthe entire gastrointestinal tract.

In one embodiment, one lipase enzyme of the combination has a pH optimumat a pH value in the range of pH ≥1 and ≤6.

In one embodiment, one lipase enzyme of the combination has a pH optimumat a pH value in the range of pH ≥8 and ≤11.

In one embodiment, the at least two lipases comprise amino acidsequences selected from the group consisting of

-   -   a) SEQ ID No 4-6, and/or fractions, variants, homologues, or        derivatives of thereof    -   b) amino acid sequences having a sequence identity of at least        70%, preferably 95% with any of SEQ ID No 4-6

These sequences relate to three preferred lipases shown in thefollowing:

TABLE 3 CILIAN pH amino AA DNA NCBI No optimum acids Sequence IDSequence ID Gene name Gene ID: 10 3.5-7   288 Seq ID No 4 Seq ID No 1TTHERM_00320120 7825111 11 6-11 288 Seq ID No 5 Seq ID No 2TTHERM_00320130 7825112 14  2-5.5 308 Seq ID No 6 Seq ID No 3TTHERM_00320230 7825120

According to another embodiment of the present invention, apharmaceutical preparation comprising the combination of two or morelipase enzymes according to the above description is provided.

According to still another embodiment of the present invention, the useof the combination of two or more lipase enzymes according the abovedescription, or of a pharmaceutical preparation comprising the latter,for

-   -   the treatment of a lipid digestion deficiency and/or a digestive        disorder, or    -   the manufacture of a medicament for the treatment of a lipid        digestion deficiency and/or a digestive disorder        is provided.

In one embodiment, in said use the digestive disorder is exocrinepancreatic insufficiency (EPI). Other digestive disorders that can betreated encompass steatorrhoea, celiac disease or indigestion EPI is theinability to properly digest food due to a lack of digestive enzymesmade by the pancreas. EPI is found in humans afflicted with cysticfibrosis and Shwachman-Diamond Syndrome, and is caused by a progressiveloss of the pancreatic cells that make digestive enzymes; loss ofdigestive enzymes leads to maldigestion and malabsorption of nutrientsfrom normal digestive processes. Chronic pancreatitis is the most commoncause of EPI in humans.

Steatorrhea is the presence of excess fat in feces. Stools may alsofloat due to excess lipid, have an oily appearance and can be especiallyfoul-smelling. An oily anal leakage or some level of fecal incontinencemay occur. There is increased fat excretion, which can be measured bydetermining the fecal fat level. The definition of how much fecal fatconstitutes steatorrhea has not been standardized.

Celiac disease is a condition in which gluten (a protein found ingrains) damages the intestinal tract. Symptoms include abdominal pain,bloating, weight loss, and fatigue. People with celiac disease mustfollow a strict diet that includes no gluten. Lipases have been studiedas part of the treatment for celiac disease, and therapy therewithresults in a modest weight gain.

Indigestion is a condition in which patients suffer bloating, gas, andfullness following a high fat meal. These symptoms are commonlyassociated with irritable bowel syndrome (MS), so some researchersspeculate that pancreatic enzymes might help treat symptoms of IBS. Nostudies have been done, however.

In one embodiment, in said use the lipid digestion deficiency isLipoprotein lipase deficiency, which is a condition caused by mutationin the gene which codes lipoprotein lipase.

According to still another embodiment of the present invention, the useof the combination of two or more lipase enzymes according the abovedescription, or of a pharmaceutical preparation comprising the latter,for the treatment of Cystic fibrosis is provided.

Cystic fibrosis is an inherited condition that causes the body toproduce abnormally thick, sticky mucus. Patients often have nutritionaldeficiencies because mucus blocks pancreatic enzymes from getting to theintestines. Taking lipases helps improve the nutrition these patientsget from food.

According to yet another embodiment, a method of producing a combinationof two or more lipase enzymes according to the above description isprovided, which method comprises the steps of

-   -   a) expressing the two or more lipase enzymes in one or more        suitable production systems, and    -   b) purifying the two or more lipase enzymes expressed in step        a).

In one embodiment, in said method at least one lipase enzyme is producedby homologous expression in an organism of the order ciliates

The term “homologous protein expression” relates to the expression of agene or protein in an organism from where said gene or proteinoriginates.

In one embodiment, said ciliate is from the family Tetrahymenidae. Morepreferably, said ciliate is from the genus Tetrahymena. Most preferably,said ciliate is from the Tetrahymena thermophile. A ciliate based lipaseproduction system provides an economical, simple and reliable method forthe production of lipases, which have a drastically increased specificactivity compared to the available competitors and thus a highlyenhanced therapeutic potential.

Since no viruses have been found in Tetrahymena combined with the greatevolutionary distance between mammalians and ciliates the safety of theproduct is expected to be much higher, while the production can be runwith more stability and less risk of failure due to viral infections.

Further, a ciliate based lipase production system is particularly usefulin case a ciliate lipase is to be produced, because ciliates have acodon usage that differs from other eukaryotes, as can be seen in thefollowing table:

TABLE 4 fields: [triplet] [frequency: per thousand] ([number]) UUU 26.1(3815) UCU 24.4 (3557) UAU 23.3 (3407) UGU 9.7 (1412) UUC 19.4 (2827)UCC 6.5 (948) UAC 14.5 (2110) UGC 8.8 (1282) UUA 29.8 (4346) UCA 16.8(2453) UAA 36.8 (5366) UGA 2.0 (286) UUG 14.1 (2054) UCG 1.5 (222) UAG11.0 (1606) UGG 7.4 (1080) CUU 20.3(2955) CCU 17.6(2574) CAU 8.7(1267)CGU 4.6(677) CUC 10.3(1497) CCC 4.6(676) CAC 6.4(930) CGC 0.9(136) CUA7.4(1078) CCA 8.2(1202) CAA 19.8(2894) CGA 0.5(73) CUG 2.6(378) CCG0.5(68) CAG 3.3(477) CGG 0.1(8) AUU 39.3(5733) ACU 27.2(3968) AAU48.0(7002) AGU 13.5(1963) AUC 16.2(2367) ACC 7.8(1140) AAC 24.2(3530)AGC 9.2(1344) AUA 19.1(2783) ACA 14.8(2153) AAA 58.7(8562) AGA26.6(3887) AUG 19.3(2811) ACG 0.8(111) AAG 34.3(5001) AGG 2.8(412) GUU25.8 (3763) GCU 30.3 (4428) GAU 42.5 (6208) GGU 24.5 (3576) GUC 10.1(1469) GCC7.5 (1098) GAC 12.4 (1815) GGC 4.3 (629) GUA 11.6 (1693) GCA11.8 (1726) GAA 58.2 (8499) GGA 15.1 (2205) GUG 3.1 (451) GCG 0.6 (88)GAG 11.2 (1630) GGG 1.5 (216) Coding GC 32.53% 1st letter GC 38.64% 2ndletter GC 31.25% 3rd letter GC 27.69%

In one embodiment, in said method at least one lipase enzyme is producedby overexpression, preferably by homologous overexpression.

The term “homologous overexpression” relates to the over-expression of agene or protein in an organism from where said gene or proteinoriginates.

For this purpose, the expression of an endogenous gene can be enhancedby external factors, e.g., it is brought under the control of a promotercloned in directly into the genome, or transcriptions factors are addedto the respective cell or organism. As an alternative, the expression ofa copy of said endogenous gene introduced into that cell or organism bymeans of a suitable plasmid can be provided. Examples for such plasmidsare shown in FIG. 6A and FIG. 6B.

According to yet another embodiment of the invention, two or morenucleic acid molecules are provided, selected from the group consistingof

-   -   a) at least one nucleic acid molecule comprising a nucleotide        sequence presented as SEQ ID NO 1-3    -   b) at least one nucleic acid molecule encoding a polypeptide        comprising the amino acid sequence presented as SEQ ID NO 4-6    -   c) at least one nucleic acid molecule that is a fraction,        variant, homologue, or derivative of the nucleic acid molecules        of a)-b),    -   d) at least one nucleic acid molecule that is a complement to        any of the nucleic acid molecules of a)-c), or capable of        hybridizing therewith under stringent conditions,    -   e) at least one nucleic acid molecule which comprises, in        comparison to any of the nucleic acid molecules of a)-d) at        least one silent single nucleotide substitution, nucleic acid        molecule according to a) and c)-e) which is code optimized for a        protozoan expression host, and/or    -   f) at least one nucleic acid molecule having a sequence identity        of at least 70%, preferably 95% with any of the nucleic acid        molecules of a)-f).

FURTHER DESCRIPTION

The inventors revealed 37 open reading frames for proteins with putativelipolytic activity in the genome of Tetrahymena. In the course of ourexperiments three enzymes were selected due to favorable properties byscreening experiments.

Tetrahymena is a nonpathogenic unicellular eukaryotic microorganismwhich has been established in a few laboratories as an expression host.It features a number of advantages which make it suitable for homologousprotein expression. Tetrahymena is a broadly examined model organism,and, in over 50 years of basic research, no viruses or endoparasiteswere observed. Examinations with indicator cell lines revealed noendogenous infectious agents like viruses or mycoplasm, which can infecthigher animals. This might be due to the nuclear dimorphism which iscommon to ciliates. Another reason for this might be the unusual codonusage and AT-rich genome in Ciliates. The inventors do thus assume thatpathogenic viruses of higher organisms cannot amplify in most ciliates.The fact that, as known so far, ciliates are not susceptible forviruses, arises as a surprising advantage. This means that in productionprocesses based on Ciliates, amplification or growth of adventitiousviruses does not occur. Furthermore it is possible to grow ciliates inanimal free media. This means, that in case a protein is produced fortherapeutic use, costly virus depletion procedures as necessary inindustrial processes with human and animal cell cultures can be skipped.

First of all, the above considerations as related to codon usage inciliates apply for Tetrahymena as well. Furthermore, high copy numberplasmids are available for Tetrahymena, containing an origin ofreplication (ori) from a minichromosomal rDNA. This minichromosomal rDNAis present in up to 9.000 copies per cell. Beyond that stableintegration can take place into the macronuclear DNA, in which all genesare present in 45-fold copy number. The high gene dose is the idealprecondition for an efficient protein biosynthesis and thus for a highproductivity. In contrast to bacteria, ciliates of the genus Tetrahymenasecrete biologically proteins very efficiently to the supernatant.

Batch, fed-batch and continuous fermentation of Tetrahymena with celldensities up to 2×10⁷ cells/ml and dry weights of up to 80 g/L areestablished, and production enlargements (upscaling) up to 1000 L couldbe demonstrated without any problem. In feasibility studies withreporter proteins space-time yields of 50-90 pg/cell a day could alreadybe achieved. First experiments with homologous expression resulted in ayield of over 200 mg/L a day for secreted proteins. Tetrahymena can befermented in conventional production facilities for microbiologicalexpression systems (bacteria or yeasts). This means that no costlymodifications in existing production plants or a new building of theproduction facilities are necessary.

Ciliate systems have, however, some other advantages with respect to theexpression of secreted enzymes. These will be discussed in thefollowing.

Despite the said advantages, ciliate expression systems are stillrelatively unknown, and the person skilled in the art, when being askedabout potential heterologous/homologous expression systems, would ratherthink of E. coli, yeast, insect cell systems (baculovirus) and mammaliancell lines.

Methods for the transformation of ciliates, which can be used in thecontext of the present invention, comprise, among others,microinjection, electroporation and particle bombardment, and are, forexample, described in Tondravi & Yao (1986), Gaertig & Gorovsky (1992)and Cassidy-Hanley et al (1997).

Methods for transformation and heterologous protein expression have beendescribed for a few protists (WO 00/58483 and WO 00/46381). Thegeneration of mitotically stable transformants of the ciliateTetrahymena thermophila can be achieved after transfection either of thesomatic macronucleus or the generative micronucleus by microinjection,electroporation or by particle bombardment.

Selection of the transformants can be performed using differentselection markers like the neomycin resistance (Weide et al. 2006, BMC)and the integration of the heterologous genes by homologous DNArecombination, which results in stable thymidin-auxotrophic Tetrahymenacells (Weide et al. 2006, BMC). In addition, the use of blasticidin S(Weide et al. 2006, BMC) or paclitaxcel (WO 00/46381) resistance hasalso been considered.

Promoters suitable for lipase expression in ciliates are, for example,disclosed in W02007006812A1 which is also registered for the applicantof the present invention, the content of which shall be incorporatedherewith by reference. Therein, a heat-inducible promoter and ametallothionein-promoter are disclosed which can also be used for thepurposes of the present invention.

Furthermore, a vector for the transfection of a ciliate host cell isprovided, said vector comprising at least one nucleic acid moleculeencoding for a lipase.

Surprisingly a combination of Tetrahymena lipases and proteases,hereinafter referred to as “the preparation”, can meet the requirementsfor the treatment of pancreatic malfunction better than any product onthe market or currently under development. Firstly, the unparalleledability of lipid digestion under various pH conditions ranging from pHvalues of 2 to pH values of up to 11 enables the preparation to digestlipids in the acidic gut and the, due to pancreatic dysfunction, acidicupper duodenum as well as in the more neutral to basic parts of thesmall intestine. Secondly, the preparation's specific activitysurprisingly was found to be at least one order of magnitude higher thanthe specific activity of pancreatin even under neutral to basicconditions. This will help to promote the patient's compliance byreducing his daily pill burden. Thirdly a predefined mixture of enzymesis contraindicated for certain forms of pathological maldigestion.

In a preferred embodiment of the present invention, two or moreTetrahymena lipases cover the physiological pH range of thegastrointestinal tract enabling lipolysis from the stomach to the lowersmall intestine. In another preferred embodiment an alkaline Tetrahymenalipase is used to digest lipids in the alkaline environment of theduodenum.

The possibility of a modular assembly of lipase, protease and amylaseactivity allows the adaptation of the preparation to patients withdifferent conditions and thus different needs of medications. Forexample high amylase content is undesirable for children withmucoviscidose.

Proteases are contraindicated in patients with acute pancreatitis oractive episodes of chronic pancreatitis. And fourthly, the preparation,in contrast to lipases from funghi is activated by bile acids inphysiologic concentrations.

Definitions

The term “ciliate”, as used herein, shall refer to the scientific phylumof Ciliophora, which are unicellular eukaryotes (“protozoa” or“protists”) characterized, among others, by their relatively large size(some species have up to 2 mm in length), their ciliated cell surfaceand by two different sorts of nuclei, i.e., a small, diploidmicronucleus, and a large, polyploid macronucleus (used for proteinexpression). The latter is generated from the micronucleus byamplification of the genome and heavy editing.

The term “cDNA”, as used herein, shall refer to a DNA molecule whichencodes for a protein to be expressed, and is devoid of any non-encodingparts, like introns. In many cases, a cDNA has been directly synthesizedfrom an mRNA template using reverse transcriptase, and an oligodT-primer. However, the term shall as well comprise synthetic genes andencoding DNAs otherwise obtained.

The term “promoter”, as used herein, shall refer to a regulatory regionof DNA generally located upstream (towards the 5′ region of the sensestrand) of a gene or a cDNA, that allows or even enhances transcriptionof the gene, or the cDNA.

The term “fragment”, as used herein, shall refer to a part of a proteinwhich lacks some parts, or domains, of the native, or wildtype proteinwhile retaining some activity in terms of enzymatic activity,immunogenity, target binding or the like.

The term “signal sequence”, as used herein, shall refer to a nucleicacid sequence which encodes for an oligopeptide (“signal peptide”) whichdirects proteins synthesized in the cytosol to certain organelles suchas the nucleus, mitochondrial matrix, endoplasmic reticulum,chloroplast, apoplast and peroxisome. Some signal peptides are cleavedfrom the protein by signal peptidase after the proteins are transported.In a stricter sense, the signal sequence, or the signal peptide,accounts for the secretion of the said protein into the exterior medium.This process takes place via the rough endoplasmic reticulum, the Golgiapparatus and subsequent exocytosis. In many cases a signal sequence islocated at the N-terminus of the protein to be secreted.

The term “operably linked” as used herein, means that a nucleotidesequence, which can encode a gene product, is linked to a promoter suchthat the promoter regulates expression of the gene product underappropriate conditions. Two nucleotide sequences that are operablylinked contain elements essential for transcription, including, forexample, a TATA box.

The term “nucleic acid molecule” is intended to indicate any single- ordouble stranded nucleic acid molecule comprising DNA (cDNA and/orgenomic DNA), RNA (preferably mRNA), PNA, LNA and/or Morpholino.

The term “stringent conditions” relates to conditions under which aprobe will preferably hybridize to its target subsequence and much lessto other sequences. Stringent conditions are sequence-dependent and willbe different in different circumstances. Longer sequences hybridizespecifically at higher temperatures. Generally, stringent conditions areselected to be about 5° C. lower than the thermal melting point (Tm) forthe specific sequence at a defined ionic strength and pH. The Tm is thetemperature (under defined ionic strength, pH and nucleic acidconcentration) at which 50% of the probes complementary to the targetsequence hybridize to the target sequence at equilibrium. (As the targetsequences are generally present in excess, at Tm, 50% of the probes areoccupied at equilibrium). Typically, stringent conditions will be thosein which the salt concentration is less than about 1.0 M Na ion,typically about 0.01 to 1.0 M Na ion (or other salts) at pH 7.0 to 8.3and the temperature is at least about 30° C. for short probes (e.g. 10to 50 nucleotides) and at least about 60° C. for longer probes.Stringent conditions may also be achieved with the addition ofdestabilizing agents, such as formamide and the like.

The term “fragment of the nucleic acid molecule” is intended to indicatea nucleic acid comprising a subset of a nucleic acid molecule accordingto one of the claimed sequences. The same is applicable to the term“fraction of the nucleic acid molecule”.

The term “variant of the nucleic acid molecule” refers herein to anucleic acid molecule which is substantially similar in structure andbiological activity to a nucleic acid molecule according to one of theclaimed sequences.

The term “homologue of the nucleic acid molecule” refers to a nucleicacid molecule the sequence of which has one or more nucleotides added,deleted, substituted or otherwise chemically modified in comparison to anucleic acid molecule according to one of the claimed sequences,provided always that the homologue retains substantially the samebinding properties as the latter.

The term “sequence identity of at least X %”, as used herein, refers toa sequence identity as determined after a sequence alignment carried outwith the family of BLAST algorithms (particularly megablast,discontiguous megablast, blastn, blastp, PSI-BLAST, PHI-BLAST, blastx,tblastn and tblastx), as accessible on the respective internet domainprovided by NCBI.

The term “vector”, as used herein, refers to a molecular vehicle used totransfer foreign genetic material into another cell. The vector itselfis generally a DNA sequence that consists of an insert (gene ofinterest) and a larger sequence that serves as the “backbone” of thevector. The purpose of a vector to transfer genetic information toanother cell is typically to isolate, multiply, or express the insert inthe target cell. Vectors called expression vectors (expressionconstructs) specifically are for the expression of the transgene in thetarget cell, and generally have a promoter sequence that drivesexpression of the transgene. Simpler vectors called transcriptionvectors are only capable of being transcribed but not translated: theycan be replicated in a target cell but not expressed, unlike expressionvectors. Transcription vectors are used to amplify their insert.

The term “plasmid”, as used herein, refers to Plasmid Vectors, i.e.circular DNA sequences that are capable of automatically replicating ina host cell. Plasmid vectors comprise an origin of replication (“ORI”)that allows for semi-independent replication of the plasmid in the hostcell. Furthermore, a plasmid may comprise a multiple cloning site whichincludes nucleotide overhangs for insertion of an insert, and multiplerestriction enzyme consensus sites to either side of the insert, apromoter to drive transcription of the plasmid's transgene, optionallyat least one genetic marker for confirmation that the plasmid hasintegrated with the host genomic DNA, and, optionally, a reporter foridentification of which cells have been successfully transfected.

The term “host cell”, as used herein, has two different meanings whichmay be understood according to the respective context. In the context ofhomologous protein expression, the term “host cell” refers to a cellwhich is used as expression host. Said cell, or its progenitor, has thusbeen transfected with a suitable vector comprising the cDNA of theprotein to be expressed.

As used herein, the term “ciliate host cell” shall refer to a cell fromthe phylum Ciliophora (formerly: Ciliata), e.g., protozoanscharacterized by the presence of hair-like organelles called cilia and anuclear dimorphism.

As used herein, the term “incorporated” shall refer to the fact that thesaid nucleic acid has entered the host cell in such way that it is readyfor protein expression. Such incorporation can have different types inciliates, e.g. “episomal incorporation” (e.g. the nucleic acid molecule,like a plasmid, has not entered the cellular nucleus, but replicates,and is translated, in the cytoplasm), and “integrative incorporation”(e.g. the nucleic acid molecule has integrated into the cellulargenome).

Disclaimer

To provide a comprehensive disclosure without unduly lengthening thespecification, the applicant hereby incorporates by reference each ofthe patents and patent applications referenced above.

The particular combinations of elements and features in the abovedetailed embodiments are exemplary only; the interchanging andsubstitution of these teachings with other teachings in this and thepatents/applications incorporated by reference are also expresslycontemplated. As those skilled in the art will recognize, variations,modifications, and other implementations of what is described herein canoccur to those of ordinary skill in the art without departing from thespirit and the scope of the invention as claimed. Accordingly, theforegoing description is by way of example only and is not intended aslimiting. The invention's scope is defined in the following claims andthe equivalents thereto. Furthermore, reference signs used in thedescription and claims do not limit the scope of the invention asclaimed.

BRIEF DESCRIPTION OF THE EXAMPLES AND DRAWINGS

Additional details, features, characteristics and advantages of theobject of the invention are disclosed in the subclaims, and thefollowing description of the respective figures and examples, which, inan exemplary fashion, show preferred embodiments of the presentinvention. However, these drawings should by no means be understood asto limit the scope of the invention.

EXAMPLES AND FIGURES

1. Construction of Expression Vectors

The genes for the different Lipases (SEQ ID No: 1, SEQ ID No: 2 and SEQID No: 3)) were cloned into the donor vector (see FIG. 6A). Theexpression cassettes from all donor vectors were transferred into theacceptor vector (see FIG. 6B) using a Cre dependent recombinase system.Sequences are given below.

2. Cultivation of Wildtype Tetrahymena and Transformation of ExpressionPlasmids (Biolistic Bombardment)

Tetrahymena thermophila strains B 1868/4, B 1868/7 and SB 1969 werecultivated in in SPPR (2.5% proteose peptone, 1% Peptone acidhydrolysate, 0.5% yeast extract, 0.1% ferrous sulphate chelate solutionand 0.2% glucose). We used conjugating T. thermophila strains. Thetransformation of the T. thermophila cells was performed as previouslydescribed in Cassidy-Hanley et al. 1997.

3. Determination of Lipase Activity

Transformed Tetrahymena clones were cultivated in SPPR medium by theaddition of 400 μg/ml paromomycin at 30° C. in a 500 ml Multifermenter.Target gene expression was induced by addition of 0.55 μM Cd²⁺ (MTT1) atthe beginning of the cultivation or in early or mid log phase. Aliquotsof cell free SPPR supernatants were harvested about 20 h to 25 h afterinduction of the culture. Lipase activity of supernatants was determinedby the colorimetric determination of liberated fatty acids described byNixon & Chang (1979), or by titration (United States Pharmacopeia 23,NF18 1095, pp 1150-1151). The screening for lipolytic active clones wasdone by a Rhodamine fluorescence test (Jette & Ziomek, 1994).

4. pH Spectra of Different Tetrahymena Lipases (FIGS. 1-3 )

The lipolytic activity of three different overexpressed Tetrahymenalipases was tested at different pH values with the Nixon test. Assubstrate a high fat pig diet from Arie Blok (Woerden, NL) which waspredigested with Pepsin at low pH to simulate gastric passage was used.Lipases No. 10 and 14 (SEQ ID NOs 4, and 6, respectively) showedactivities at low pH values while lipase no. 11 exhibited a broad pHactivity spectrum from neutral to high pH values comparable toPancreatin. A combination of these lipases can cover pH values from 2 to11. Results are shown in FIGS. 1-3 .

5. Stability in Gastric Juice (FIG. 4 )

pH activities were determined after incubation for 0.5 and 3 h in humangastric fluid (FIG. 4 ). In contrast to Pancreatin derived productslipases No. 10 and 14 retain activities of 38% and 30% respectively evenafter 3 h of incubation in human gastric fluid. Results are shown inFIG. 4 .

6. Bile Salt Activation (FIG. 5 )

The lipolytic activity of lipase No. 11 (SEQ ID No 5) was tested in thepresence of various amounts of a physiologic mixture of bile acids(Gargouri et al., 1986). Like human pancreas lipase, lipase No. 11 isactivated by increasing concentrations of bile acids. Results are shownin FIG. 5 .

Sequences SEQ ID No 1: Nucleotide of Lipase 10    1atgaaattgt aattgcttct attggtttgc ttgtcatttg ctgcctgcta atcatttact   61tatacttaat cacttgctta agacttagct ggtttctctc ttgcttctta ctgtaatcct  121aaatctatag aacaatggaa ttgtggatgt gcttgtgata aaaaccctta aggacttcga  181aatgttacta tcttatttaa ctctactcta taagctagtg gatatttagg ctactccact  241catcatgatg caattgttgt tgtattcaga ggaacagtac cttggttaat cgaaaattgg  301attgctgact taaacacctt caagacttag tacccactct gccaaaactg ttatgtccat  361taaggctttt ataaccagtt caaataattg aaatctcagc ttgttactag ctttacttca  421cttcgttaac tatatcctaa tgcaaaagta tttgttacag gacattctct tggtgctgca  481atgagtgctc actcaatacc agtaatttac taattaaatg gaaataaacc tattgatgct  541ttttacaatt atggttgtcc tagagtaggt gactaaactt atgcaaactg gtttaacagt  601taaaattttg ccttagaata tggtagaatt aataatgctg ctgatccagt tcctcattta  661cctcctcttc tttacccatt ttcatttttc cactacaacc atgaaatatt ctatccttct  721tttgttcttt ttggaaacta acataactaa tgttaaaacg cggaaacaat atttggtgca  781gatggagtaa taatagcagc taatgttcta gaccatctaa cttattttgg atgggattgg  841tctggttcta tattaacttg ctaatgaSEQ ID No: 4: amino acid sequence of Lipase 10MKLQLLLLVCLSFAACQSFTYTQSLAQDLAGFSLASYCNPKSIEQWNCGCACDKNPQGLRNVTILFNSTLQASGYLGYSTHHDAIVVVFRGTVPWLIENWIADLNTFKTQYPLCQNCYVHQGFYNQFKQLKSQLVTSFTSLRQLYPNAKVFVTGHSLGAAMSAHSIPVIYQLNGNKPIDAFYNYGCPRVGDQTYANWFNSQNFALEYGRINNAADPVPHLPPLLYPFSFFHYNHEIFYPSFVLFGNQHNQCQNAETIFGADGVIIAANVLDHLTYF GWDWSGSILTCQSEQ ID No: 2: Nucleotide of lipase 11    1atgaaatcaa tttttttatt aattatttcc ttgcttttag cttcttgctc atagttttaa   61tataatgaaa cacttgccta agacttagct ggattttctc ttgcttctta ctgtaatcct  121aaatatttat aataatggaa ttgtggctct gcttgtaaaa aaaacccaaa tggtcttaca  181gatttctctt atttgtataa caagacttta aaggcaagtg gatatatagg ctattctgct  241catcatgatg ctattatagt tgtctttaga ggaactgtcc cttggttgat ctaaaattgg  301attgcagatt taaacactat caaaatttaa tatcctttct gtgaaaattg ttatgttcat  361aaaggtttct ataaatagtt caattaatta aaatcttaac ttatttaaag ctttacagaa  421attcgttaaa aatatccttc atcaaaaata tttgtcactg gacattctct tggtgcagct  481atgagttttc attcaatgcc tattattttt gaattaaatg gaaataagcc tattgatgct  541ttctataatt atggttcccc aagagttggt aacgaagcat atgcaacttg gtttaattta  601caaaattttg ctttataata tggcagaata aataatgcag cagatcctgt tcctcattta  661cctcctattc ttttcccttt ctaattttat catactaatc atgaaatatt ttatacttca  721tttattgaag atggtaacaa atatgagtaa tgcttagatg cagaacacaa attatgtgca  781aatagtaaga ttattgctgc aagcgttcgt gaccatctta gttattttgg ctggaattgg  841gctacttcta ttttaacttg ccaatgaatt aaaaaattaa tttatcaaac aaaaacatta  901actaaaatta tttttatctg tttaaatttg ttttaaaaca tttatatat attttaatat  961ttactacttt ttagaataaa atatctSEQ ID No: 5: Amino acid sequence of lipase 11MKSIFLLIISLLLASCSQFQYNETLAQDLAGFSLASYCNPKYLQQWNCGSACKKNPNGLTDFSYLYNKTLKASGYIGYSAHHDATIVVFRGTVPWLIQNWIADLNTIKIQYPFCENCYVHKGFYKQFNQLKSQLIQSFTEIRQKYPSSKIFVTGHSLGAAMSFHSMPIIFELNGNKPIDAFYNYGSPRVGNEAYATWFNLQNFALQYGRINNAADPVPHLPPILFPFQFYHTNHEIFYTSFIEDGNKYEQCLDAEHKLCANSKIIAASVRDHLSYF GWNWATSILTCQSEQ ID No: 3: Nucleotide acid sequence of lipase 14    1ATGAACAAAT TGCAAGTTCT TTTCATTGCA GCTATAGTTT GCACAATTGG ATCCACTGTT   61TATTTACTCA ATAAGAGCTC TTCAGATGTC CAAGAGTCTT AACTGACTTT CCCCTATGAT  121GAAAATTTAG CTGAAAATTT AGCTGGATTT TCTATGGCTT CTTATTGTAAAGCTTCTAAA  181ATTGAAAACT GGAATTGCGG TGCTTCTTGC AAAAAAAATC CCGAAGGACT TTAAGATGTC  241TACATTATGA AAAATAAAAC TATGAACGCT GCTGGTTTCT TAGCATATTC TCCTGCTCAT  301GATGCTATAG TAGTTGTATT TAGAGGAACT GTCCCCTGGT TGATCAAGAA TTGGATTAGT  361GACATTAACA CTGTCAAAAC AAAATACTCT AGATGCGAAA AATGCTATGT TCATTTGGGC  421TTCTTCAATG CCTTCAAGGA ATTGTAAGAT TAAATTCTTA CTGAGTTCCC TAAACTTAAG  481GCCAAATATC CTTATTCAAA GGTAATTTAA CACAAAATAT ACATATATCT CTTTATAAAT  541AATTCATGCT ATCATATGTT TTTCTTTAGA TTATTGTGTA TTTCAAAAGC ATCACCTTAG  601CCTTTAAATA TTGATTAAGG AAATATTAAA TGATTTGTAA AATCAATTGC AAGAATATAA  661ATTACTCTAA ATTAAATCGA CGTATGAATC GAATACCCAA CTAATTATAG GCATTAATAA  721ATTTTGGAAA ATTATTTGTT TTCTCAATTT TCAATATGAA AATTTAGCTT AACTTATTTG  781GCTTTTAATA TTTATTCCAC TTTTTACATC TTATTCATCA ATTATATTTA TTTTAAACTC  841ATTTAAAAAT AAATAGGTTT TTGTTACAGG TCATTCCCTT GGTGCTGCAA TGAGTACTCA  901CGCTGTTCCT GTCATTTATG AACTCAATGG AAATAAGCCT ATCGATGCAT TCTATAATTT  961TGGTTCCCCT AGGGTTGGTG ATGAAAATTA CCACTAATGG TTCGATAGCT AAAATTTTAC 1021TCTTTAATAT GGTAGAATTA ACCACAGAGC TGATCCAGTT CCTCATTTAC CCCCTAATTA 1081CTCTCCTTTC ACTTTTACTC ATATTGATCA TGAAGTTTTC TATTAAACAT TTAAGAAACC 1141TTATACATAA TGTATTGAAA CTGAAAGTCT TGAATGTGCT GATGGTATAAAAATTCCCTT 1201AGATATTCCT GACCATCTTT CTTACTTTGG TTGGGATTGG GCCACTGACA TCTTAGCTTG 1261CTAATGA SEQ ID No: 6: amino acid sequence of lipase 14MNKLQVLFIAAIVCTIGSTVYLLNKSSSDVQESQLTFPYDENLAENLAGFSMASYCKASKIENWNCGASCKKNPEGLQDVYIMKNKTMNAAGFLAYSPAHDAIVVVFRGTVPWLIKNWISDINTVKIKYSRCEKCYVHLGFFNAFKELQDQILTEFFKLKAKYPYSKVFVTGHSLGAAMSTHAVPVIYELNGNKPIDAFYNFGSPRVGDENYHQWFDSQNFTLQYGRINHRADPVPHLPPNYSPFTFTHIDHEVFYQTFKKPYTQCIETESLECADGIKIPLDIPDHL SYFGWDWATDILACQ

REFERENCES

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What is claimed is:
 1. A pharmaceutical preparation comprising acombination of two or more lipase enzymes, wherein: at least one lipaseenzyme has a pH optimum at a pH value in the range of ≥1 and ≤6; atleast one other lipase enzyme has a pH optimum at a pH value in therange of ≥8 and ≤11; and the two or more lipase enzymes comprise aminoacid sequences having a sequence identity of at least 70% with any ofSEQ ID Nos: 4-6.
 2. The pharmaceutical preparation of claim 1, whereinlipolytic activity of at least one lipase enzyme is determined with aNixon Test or a titration test.
 3. The pharmaceutical preparation ofclaim 1, wherein at least one lipase enzyme is a lipase enzyme encoded,expressed and/or produced by an organism of the order ciliates.
 4. Thepharmaceutical preparation of claim 1, wherein at least one lipaseenzyme has been modified by site directed or random mutagenesis andsubsequent selection.
 5. The pharmaceutical preparation of claim 1,wherein one lipase enzyme has a pH optimum at an acidic pH which occursin the stomach of a mammal and wherein at least one other lipase enzymehas a pH optimum at an alkaline pH which occurs in the lower smallintestine of a mammal.
 6. The pharmaceutical preparation of claim 1,wherein the two or more lipase enzymes comprise amino acid sequencesselected from the group consisting of amino acid sequences having asequence identity of at least 95% with any of SEQ ID Nos: 4-6.